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H9N2 subtype avian influenza virus-duck enteritis virus living-vector vaccine

A technology for avian influenza virus and duck enteritis virus, which is applied in the directions of virus/bacteriophage, antiviral agent, antibody medical component, etc., can solve the problems of difficult operation of avian influenza vaccine strains, and achieve the effect of shortening the cycle.

Inactive Publication Date: 2014-06-25
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is difficult to construct H5 and H9 subtype avian influenza vaccine strains using traditional methods. Using genetic manipulation methods such as bacterial artificial chromosome technology, a recombinant duck enteritis virus live vector vaccine containing the ha gene of influenza virus is constructed to provide a source for the development of poultry vaccines. new technical means

Method used

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  • H9N2 subtype avian influenza virus-duck enteritis virus living-vector vaccine
  • H9N2 subtype avian influenza virus-duck enteritis virus living-vector vaccine
  • H9N2 subtype avian influenza virus-duck enteritis virus living-vector vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0051] The construction of embodiment 1BAC homology arm and the formation of multiple copies

[0052] 1. Extraction of duck viral enteritis (DEV) virus genome

[0053] The frozen duck enteritis virus vaccine strain C-KCE (this biological material has been published, see literature 16 (Chen et al.Wei Zou et al.

[0054] Construction of a full-length infectious bacterial artificial chromosome clone of duck enteritis virus vaccine strain. Virology Journal 2013, 10:328) its genome DNA sequence has been submitted, see GenBank ID: KF263690. The strain was collected from a duck farm in Xianning City, Hubei Province, China in 2010 by the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University. The preparation method is as follows: add 0.9% normal saline to the collected duck farm disease material, homogenate and inoculate chicken embryos with the supernatant after centrifugation, collect chicken embryo allantoic fluid after blind passage for three generati...

Embodiment 2

[0060] Screening and purification of embodiment 2 recombinant virus

[0061] 1. Preparation of CEF cells

[0062] Take 9-10-day-old SPF chicken embryos (purchased from Beijing Meria Weitong Experimental Animal Technology Co., Ltd.), sterilize them with alcohol cotton balls, wipe the gas chamber with tincture of iodine for deiodination, take out the chicken embryos aseptically, and place them in a sterile environment. The bacteria were placed in glass vials, and the head, limbs and viscera were removed and chopped with ophthalmic scissors. Use sterilized phosphate buffer saline (referred to as PBS, formula: Na 2 CO 3 1.59g,NaHCO 3 2.93g, with ddH 2 Dilute the volume to 1000ml, adjust the pH to 7.4) Wash twice in the bacterial bottle, add an appropriate amount of 0.25% trypsin to digest in a water bath at 37°C for 30min, suck and discard the trypsin, and use an appropriate amount of fetal bovine serum (10%) with Double antibiotics (penicillin 100u / mL, streptomycin 100u / mL) ...

Embodiment 3

[0067] Example 3 Electric transfer of recombinant virus into Escherichia coli DH10B-IS

[0068] 1. Preparation of competent DH10B-IS: Streak Escherichia coli (DH10B-IS, gifted by Professor Ma Lixin, School of Life Sciences, Hubei University) stored at -80°C on LB plates, and culture overnight at 37°C. Insert the single colony of activated Escherichia coli into LB or add the corresponding antibiotics (chloramphenicol (Cam), 34pg / mL, streptomycin (Str) 50pg / mL) in the LB liquid medium, 37℃200~250r / mL Min shaking overnight transfer 0.2 ~ 1mL overnight culture to Erlenmeyer flask filled with 200mL LB. Incubate vigorously at 37°C for 2 to 6 hours, and measure OD regularly 600 value. When OD 600 When the value reaches 0.7-1.0, take out the Erlenmeyer flask from the shaker, place it in an ice-water mixture, and bathe in ice for 15 minutes. Collect the bacteria in a pre-cooled sterile centrifuge tube, centrifuge at 6000r / min at 4°C for 10min, and discard the supernatant. Resuspend ...

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Abstract

The invention belongs to the technical field of animals genetic engineering, in particular relates to an application of H9N2 subtype avian influenza virus-duck enteritis virus living-vector vaccine strain rDEV-HA9 in constructing a living-vector vaccine. The vaccine strain is preserved in the China Center for Type Culture Collection( the preservation number is CCTCC NO: V201403). The construction method of the living-vector vaccine disclosed by the invention comprises inserting H9N2 subtype avian influenza virus ha gene fragment in the duck enteritis virus artificial chromosome plasmid pBAC-C-KCE to obtain a recombinant plasmid pBAC-C-KCE-HA9; wherein the genetic structural compositions of the plasmid pBAC-C- KCE are shown in Figure 7 and the genetic structural compositions of plasmid pBAC-C-KCE-HA9 are shown in Figure 15.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and in particular relates to the construction and application of a H9N2 subtype avian influenza virus and duck enteritis virus live vector vaccine. Background technique [0002] Duck Virus Enteritis (DEV), also known as duck plague (Duck Plague, DP), is an acute, contact infection of ducks, geese and other birds of the order Anseriformes caused by Duck Enteritis Virus (DEV). Sexually transmitted diseases can occur in poultry of all ages. Duck viral enteritis has been reported in many countries, and it is also widely prevalent in our country. Due to its rapid spread, high morbidity and mortality rate of 50-100%, and even more than 90% of adult ducks, it has become one of the main diseases that endanger the duck industry. In recent years, there have been successive research reports on the genome of duck enteritis virus; in 2009, the genome sequence of duck enteritis virus had b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/295C12N1/21A61K39/145A61K39/245A61P31/16A61P31/22C12R1/93
CPCY02A50/30
Inventor 金梅林李淑云邹忠
Owner HUAZHONG AGRI UNIV
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